A protoplast transformation system has been developed for Corynebacterium glutamicum by using a C. glutamicum-Bacillus subtilis chimeric vector. The chimera was constructed by joining a 3.0-kilobase cryptic C. glutamicum plasmid and the B. subtilis plasmid pBD10. The neomycin resistance gene on the chimera, pHY416, was expressed in C. glutamicum, although the chloramphenicol resistance gene was not. The various parameters in the transformation protocol were analyzed separately and optimized. The resulting transformation system is simple and routinely yields 10(4) transformants per microgram of plasmid DNA.